Senescence-associated Secretory Phenotype Factors Research Articles

(-)-α-Bisabolol inhibits D-Gal-induced HSF cellular senescence in vitro and prevents skin aging in vivo by reducing SASP.

To study the anti-aging effect of (-)-α-bisabolol ((-)-α-bis) on the skin and preliminarily clarify its mechanism. Human skin fibroblasts (HSF) were induced senescence by D-Galactose. Senescence β-galactosidase staining was utilized to evaluate the senescence of HSF. TNF-α, IL-6, IL-8, IL-1β, CCL-2, CCL-5, and MMP-9 in senescence-as-sociated secretory phenotype (SASP) were detected by RT-qPCR. Meanwhile, aged BALB/c mice were applied topically with 0.5% and 2%(-)-α-bis gel for 30 days continuously to evaluate anti-aging parameters on the skin such as surface measurement, the Trans Epidermal Water Loss (TEWL), and skin barrier index of dorsal skin. Then, HE staining, Masson staining, and IHC were applied to measure epidermal thickness, collagen fiber content in the dermis, and content of dermal collagen I, respectively. Last, SOD, MDA, and HYP contents of the back skin tissue of mice were also detected. (-)-α-Bis reduced the expression of senescence-associated β-galactosidase (SA-β-gal) and expression levels of SASP in HSF cells stimulated by D-Gal (P<0.05). Mice aged 9 months were applied locally with (-)-α-bis gel to improve skin aging, the TEWL and skin barrier index of dorsal skin, and ameliorate the epidermal thickness and contents of dermal collagen fibers and collagen I (P<0.05). Furthermore, (-)-α-bis up-regulated the mRNA expression levels of elastin and collagen III effectively (P<0.05). (-)-α-Bis can delay the senescence of HSF cells by reducing the expression of SA-β-gal and SASP factors in vitro. Improved skin barrier function as well as SASP is responsible for the delay of skin aging in vivo.

NAD+ associated genes as potential biomarkers for predicting the prognosis of gastric cancer.

Nicotinamide adenine dinucleotide (NAD+) plays an essential role in cellular metabolism, mitochondrial homeostasis, inflammation, and senescence. However, the role of NAD+-regulated genes, including coding and long non-coding genes in cancer development is poorly understood. We constructed a prediction model based on the expression level of NAD+ metabolism-related genes (NMRGs). Furthermore, we validated the expression of NMRGs in gastric cancer (GC) tissues and cell lines; additionally, β-nicotinamide mononucleotide (NMN), a precursor of NAD+, was used to treat the GC cell lines to analyze its effects on the expression level of NMRGs lncRNAs and cellular proliferation, cell cycle, apoptosis, and senescence-associated secretory phenotype (SASP). A total of 13 NMRGs-related lncRNAs were selected to construct prognostic risk signatures, and patients with high-risk scores had a poor prognosis. Some immune checkpoint genes were upregulated in the high-risk group. In addition, cell cycle, epigenetics, and senescence were significantly downregulated in the high-risk group. Notably, we found that the levels of immune cell infiltration, including CD8 T cells, CD4 naïve T cells, CD4 memory-activated T cells, B memory cells, and naïve B cells, were significantly associated with risk scores. Furthermore, the treatment of NMN showed increased proliferation of AGS and MKN45 cells. In addition, the expression of SASP factors (IL6, IL8, IL10, TGF-β, and TNF-α) was significantly decreased after NMN treatment. We conclude that the lncRNAs associated with NAD+ metabolism can potentially be used as biomarkers for predicting clinical outcomes of GC patients.

Flavonoid 4,4′-dimethoxychalcone selectively eliminates senescent cells via activating ferritinophagy

Flavonoids are bioactive natural polyphenolic compounds with health benefits, including anti-tumor, anti-inflammatory and anti-aging effects. Our previous studies revealed that a flavonoid 4,4′-dimethoxychalcone (DMC) induced ferroptosis via inhibiting ferrochelatase (FECH). However, the effect of DMC on cellular senescence is unknown. In the present study, we found that DMC treatment selectively eliminated senescent cells, and DMC alone or a combination of DMC and quercetin or dasatinib showed high efficiency in the clearance of senescent cells. We identified FECH was highly expressed in senescent cells compared to non-senescent cells. Mechanistically, we found that DMC inhibited FECH and induced ferritinophagy, which led to an increase of labile iron pool, triggering ferroptosis of senescent cells. Importantly, we found that DMC treatment prevented hair loss, improved motor coordination, and reduced the expression of several senescence-associated secretory phenotype factors (IL-6, IL-1β, CXCL-10, and MMP12) in the liver of old mice. Collectively, we revealed that, through the induction of ferroptosis, DMC holds the promise as a new senolytics to prevent age-related pathologies.

ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD), which still lacks of reliable markers and therapeutic options. Cellular senescence has been considered an important mechanism of intestinal fibrosis, but the underlying molecular link remains elusive. Tissues were stained using α-smooth muscle actin (α-SMA), fibronectin, and collagen I as markers of myofibroblastic differentiation. Cellular senescence was confirmed through Lamin B1 staining, senescence-associated β-galactosidase staining, and the expression of senescence-associated secretory phenotype (SASP) factors. We explored the relationship between senescence of intestinal epithelial cells (IECs) and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. The effects of irisin on cellular senescence and fibrosis were determined. Here, we identify engulfment and cell motility protein 1 (ELMO1) as a novel biomarker for intestinal cellular senescence and fibrosis. In fibrostrictured tissues from patients and murine models with IBD, significantly high levels of cellular senescence score and factors were noted, which positively correlated with the fibrotic regulator fibronectin. Senescent IECs, not fibroblast itself, released SASP factors to regulate fibroblast activation. Prolonging exposure to severe and persistent injurious stimuli decreased ELMO1 expression, which dampened SIRT1 deacetylase activity, enhanced NF-κB (p65) acetylation, and thereby accelerated cellular senescence. Deletion of ELMO1 led to senescent IECs accumulation and triggered premature fibrosis in murine colitis. Furthermore, irisin, inhibiting the degradation of ELMO1, could downregulate p65 acetylation, reduce IECs senescence, and prevent incipient intestinal fibrosis in murine colitis models. This study reveals ELMO1 downregulation is an early symbol of intestinal senescence and fibrosis, and the altered ELMO1-SIRT1-p65 pathway plays an important role in intestinal cellular senescence and IBD-related fibrosis.

CIRCULATING SASP FACTORS ARE ELEVATED IN PATIENTS WITH POSTOPERATIVE DELIRIUM

Abstract Aging, a major risk factor for delirium, is associated with accumulation of senescent cells in many tissues of the body. Senescent cells produce a host of proinflammatory and tissue remodeling factors collectively termed the senescence-associated secretory phenotype (SASP), which are secreted into circulation. The relationship between increased senescent cell burden and risk of delirium is not known. This study aimed to determine whether changes in circulating SASP proteins are associated with increased risk of postoperative delirium in cognitively intact older adults. Participants were recruited among patients scheduled for surgical or transcatheter aortic valve replacement, and blood was obtained at the presurgical visit. SASP factors were measured using commercially available multiplex magnetic bead immunoassays based on Luminex xMAP multianalyte profiling platform and analyzed on a MAGPIX system. Activin A concentration was determined by a Quantikine ELISA kit. Delirium cases were identified by manual chart abstraction, utilizing the problem list, ICD-10 codes, clinical notes, and nursing flowsheets. Eight of 62 (12.9%) of patients within the cohort developed postoperative delirium. Length of stay was significantly longer for patients who developed delirium (10.6 days) versus patients without delirium (6 days). Concentrations of SASP factors Activin A and PAPP-A were significantly higher in plasma of patients with delirium vs. those who did not develop delirium. IL-1 alpha concentration was significantly lower in patients with delirium vs without delirium. Other SASP factors measured were not statistically different between groups. More study is needed to determine whether these changes are directly related to increased senescent cell burden.

Counteracting diabetes-induced adipose tissue derived-stromal cell senescence

Adipose tissue stromal cells (ADSCs) are prone to functional decline and senescence during metabolic disturbances. In diabetes mellitus (DM), the pathogenic microenvironment induces oxidative stress causing ADSCs to senesce. The senescence associated secretory phenotype (SASP) in turn drives disease progression. The pathogenesis of DM is thus both a cause and consequence of senescence. Therapeutically preventing the onset of senescence in ADSCs may play a significant role in preventing disease progression and directly impact the onset of comorbidities. The purpose of this study was to establish an in vitro model that mimic the DM micro-environment to use as a screening tool to assess the therapeutic efficacy of preventative and restorative agents. Exposing ADSCs (<passage 10) to a combination of high glucose, advanced glycation end products (AGE-BSA) and TNFα in culture for a period of 3 days induced senescence in 70 ± 4 % of cells (β-galactosidase assay). This coincided with increased ROS production, DNA damage (yH2Ax foci) and excessive release of SASP factors (IFNγ, TNFα, IL1β, IL8, IL6). Pretreatment of ADSCs as well as early intervention using either Metformin (50 μg/mL) or ascorbic acid 2 phosphate (AAP 0.6 mM) could prevent the onset of premature senescence. The expression of genes related to cell cycle (p21cip1, p16INK4A), apoptosis (p53), inflammation (TNFα, IL6, PTX3, IL10) and adipogenesis (PPARγ, UCP3) was assessed, with AAP pretreatment significantly reducing PTX3 expression. Although this study only investigated the early stages following senescence induction (3 days), subsequent studies could employ this model for longer duration and gain insight into premature senescence and the functional decline of ADSCs within the diabetic context, whilst limiting the use of animals.

Attenuation of pro-tumorigenic senescent secretory phenotype by StN, a novel derivative of stevioside, potentiates its inhibitory activity on hepatocellular carcinoma

Ent-13-Hydroxy-15-kaurene-19-acid N-Methylpiperazine Ethyl Ester (StN) is a novel derivative of the natural diterpene stevioside isolated from Stevia rebaudiana (Bertoni). In this study, we examined the effects of StN against hepatocellular carcinoma (HCC) in vitro and in vivo as well as its anticancer mechanisms by inhibiting proliferation and regulating the senescence-associated secretory phenotype (SASP). We showed that StN significantly inhibited HCC cell proliferation by inducing cellular senescence, as observed by increased senescence-associated β-galactosidase activity and cell cycle arrest. Mechanistically, StN impaired lysosomal stability and triggered the release of cathepsin B from the lysosomes into the nucleus where it promoted DNA damage. Cathepsin B-mediated DNA damage contributed to cellular senescence triggered by StN. Meanwhile, StN transcriptionally suppressed multiple pro-inflammatory SASP components, including IL-6, IL-1α, IL-1β, and IL-8, resulting in the reduction of pro-tumorigenic impact of SASP. Further study revealed that StN inactivated NF-κB and PI3K/Akt signaling, which significantly accounted for its inhibition on the SASP factors. In HCC xenograft mice, administration of StN significantly suppressed tumor growth, while no significant toxicity was detected. This study demonstrates a novel mechanism that suppressing the SASP by StN in senescent cells potentiates its anticancer efficacy, thus defining a potential compound for cancer treatment.

Chronic low-grade inflammation and ovarian dysfunction in women with polycystic ovarian syndrome, endometriosis, and aging.

The ovarian microenvironment is critical for follicular development and oocyte maturation. Maternal conditions, including polycystic ovary syndrome (PCOS), endometriosis, and aging, may compromise the ovarian microenvironment, follicular development, and oocyte quality. Chronic low-grade inflammation can induce oxidative stress and tissue fibrosis in the ovary. In PCOS, endometriosis, and aging, pro-inflammatory cytokine levels are often elevated in follicular fluids. In women with obesity and PCOS, hyperandrogenemia and insulin resistance induce ovarian chronic low-grade inflammation, thereby disrupting follicular development by increasing oxidative stress. In endometriosis, ovarian endometrioma-derived iron overload can induce chronic inflammation and oxidative stress, leading to ovarian ferroptosis and fibrosis. In inflammatory aging (inflammaging), senescent cells may secrete senescence-associated secretory phenotype factors, causing chronic inflammation and oxidative stress in the ovary. Therefore, controlling chronic low-grade inflammation and fibrosis in the ovary would present a novel therapeutic strategy for improving the follicular microenvironment and minimizing ovarian dysfunction.

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence.

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on-2 weeks off-1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16INK4A -positive senescent cells, we found that exvivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Exvivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

The effect of Dendrobium officinale extract in inhibiting the senescence of H2O2-induced fibroblasts based on the senescence-associated secretory phenotype.

Dendrobium officinale is widely used for a long time in China, with effect of antioxidation, antitumor, enhancing immunity and so on. In recent years, Dendrobium officinale has been gradually used in cosmetics due to its powerful beauty effects. Based on senescence-associated secretory phenotype (SASP), we studied the antiaging effect of Dendrobium officinale extract (DOE) on skin. The senescent model of human skin fibroblasts was established by the induction of H2O2, and the content of SASP factors was tested after the treatment of DOE, such as interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-1 (MMP-1). It was found that after the treatment with different concentrations of DOE, the contents of IL-6, MCP-1 and MMP-1 all decreased in different degrees. It indicated that DOE could inhibit the secretion of SASP factors and was a promising natural antiaging agent.

PDK4 rescues high-glucose-induced senescent fibroblasts and promotes diabetic wound healing through enhancing glycolysis and regulating YAP and JNK pathway

During the process of wound healing, fibroblasts migrate to the wound site and perform essential functions in promoting cell proliferation, as well as synthesizing and secreting the extracellular matrix (ECM). However, in diabetic wounds, senescent fibroblasts exhibit impaired proliferative capacity and fail to synthesize essential ECM components. Pyruvate dehydrogenase kinase 4 (PDK4), a key enzyme regulating energy metabolism, has been implicated in modulating cellular senescence and fibroblast function. However, its specific role in diabetic wounds remains poorly understood. In this study, we conducted a series of in vivo and in vitro experiments using STZ-induced diabetic mice and human dermal fibroblasts. We evaluated cellular senescence markers, including SA-β-gal, P53, P16, P21, and PAI-1, as well as senescence-associated secretory phenotype (SASP) factors. Finally, we observed that PDK4 increased in normal wound healing, but its expression was insufficient in diabetic wounds. Significantly, the overexpression of PDK4 demonstrated the potential to accelerate diabetic wound healing and improve the senescence phenotype both in vivo and in vitro. Furthermore, our study elucidated the underlying mechanism by which PDK4 improved the senescent phenotype through the enhancement of glycolysis and regulation of YAP and JNK pathway. The effect was dependent on metabolic reprogramming and subsequent reduction of reactive oxygen species (ROS), which was mediated by PDK4. Overall, our findings highlight the potential of PDK4 as a promising therapeutic target for addressing diabetic wounds.

YBX1 Regulates Satellite II RNA Loading into Small Extracellular Vesicles and Promotes the Senescent Phenotype.

Senescent cells secrete inflammatory proteins and small extracellular vesicles (sEVs), collectively termed senescence-associated secretory phenotype (SASP), and promote age-related diseases. Epigenetic alteration in senescent cells induces the expression of satellite II (SATII) RNA, non-coding RNA transcribed from pericentromeric repetitive sequences in the genome, leading to the expression of inflammatory SASP genes. SATII RNA is contained in sEVs and functions as an SASP factor in recipient cells. However, the molecular mechanism of SATII RNA loading into sEVs is unclear. In this study, we identified Y-box binding protein 1 (YBX1) as a carrier of SATII RNA via mass spectrometry analysis after RNA pull-down. sEVs containing SATII RNA induced cellular senescence and promoted the expression of inflammatory SASP genes in recipient cells. YBX1 knockdown significantly reduced SATII RNA levels in sEVs and inhibited the propagation of SASP in recipient cells. The analysis of the clinical dataset revealed that YBX1 expression is higher in cancer stroma than in normal stroma of breast and ovarian cancer tissues. Furthermore, high YBX1 expression was correlated with poor prognosis in breast and ovarian cancers. This study demonstrated that SATII RNA loading into sEVs is regulated via YBX1 and that YBX1 is a promising target in novel cancer therapy.

Salvianolic acid B protects against pulmonary fibrosis by attenuating stimulating protein 1-mediated macrophage and alveolar type 2 cell senescence.

Idiopathic pulmonary fibrosis (IPF), as the most common idiopathic interstitial pneumonia, is caused by a complex interaction of pathological mechanisms. Interestingly, IPF frequently occurs in the middle-aged and elderly populations but rarely affects young people. Salvianolic acid B (SAB) exerts antioxidant, antiinflammatory, and antifibrotic bioactivities and is considered a promising drug for pulmonary disease treatment. However, the pharmacological effects and mechanisms of SAB on cellular senescence of lung cells and IPF development remain unclear. We used bleomycin (BLM)-induced pulmonary fibrosis mice and different lung cells to investigate the antisenescence impact of SAB and explain its underlying mechanism by network pharmacology and the Human Protein Atlas database. Here, we found that SAB significantly prevented pulmonary fibrosis and cellular senescence in mice, and reversed the senescence trend and typical senescence-associated secretory phenotype (SASP) factors released from lung macrophages and alveolar type II (AT2) epithelial cells, which further reduced lung fibroblasts activation. Additionally, SAB alleviated the epithelial-mesenchymal transition process of AT2 cells induced by transforming growth factor beta. By predicting potential targets of SAB that were then confirmed by chromatin immunoprecipitation-qPCR technology, we determined that SAB directly hampered the binding of transcription factor stimulating protein 1 to the promoters of SASPs (P21 and P16), thus halting lung cell senescence. We demonstrated that SAB reduced BLM-induced AT2 and macrophage senescence, and the subsequent release of SASP factors that activated lung fibroblasts, thereby dual-relieving IPF. This study provides a new scientific foundation and perspective for pulmonary fibrosis therapy.

The senescence-associated secretome for prediction of mortality in patients with aortic valve stenosis undergoing TAVR

Abstract Background Cellular senescence is an essential mechanism in the process of aging. The senescence-associated secretory phenotype (SASP) includes mainly pro-inflammatory cytokines, proteases, soluble signalling factors, and growth modulators. Degenerative aortic valve stenosis (AVS) is the prototypical inflammatory age-related cardiovascular disease. Purpose Our aim was to assess whether circulating concentrations of SASP factors are associated with prognosis in elderly patients with AVS undergoing transcatheter aortic valve replacement (TAVR), the current standard therapy for severe cases. Methods A total of 24 SASPs were measured using the Luminex Multiplex ELISA Assay (Biotechene, GmbH) in 120 AVS patients undergoing TAVR between February 2017 and February 2020 with a prospective follow-up of up to 4 years. Receiver operating characteristic (ROC) curves were created for each SASP protein in order to identify predictive markers. SASPs were considered positive if their baseline value was above the cut-off value derived from ROC-curves. The only 3 independent predictive factors were selected to develop an easy-to-calculate score based on the number of positive SASP proteins (0 to 3). Primary endpoint was long-term all-cause mortality. Results Median (IQR) age of the population was 83.2 (78.9-87.0) years, 56.7% were women. According to ROC curves, 9 out of 24 SASPs showed a significant area under the curve (AUC), with values above 0.623 (see Table). Only 3 of the markers reflecting potentially different underlying mechanisms remained independent predictors, after including all SASP factors in a Cox Regression analysis: GDF-15, ICAM-1 and Osteoprotegerin (all, p&amp;lt;0.03). A score based on the number of baseline factors being above the AUC-derived threshold level showed an excellent discrimination ability to predict survival, with long-term all-cause mortality substantially increasing for each additional positive marker (see Kaplan-Meier curves). Conclusion In conclusion, the presence of elevated baseline levels for 0, 1, 2, or 3 of the independent SASP factors reflecting different underlying pathophysiological mechanisms predicts increased long-term all-cause mortality with profoundly improved discrimination power in AVS patients undergoing TAVR. If confirmed in further prospective studies, a combination of these 3 SASP markers could serve as a highly useful risk stratification tool in the elderly population with AVS.

Spontaneous p53 activation in middle-aged C57BL/6 mice mitigates the lifespan-extending adaptive response induced by low-dose ionizing radiation

Understanding the biological effects of low-dose (<100 mGy) ionizing radiation (LDR) is technically challenging. We investigated age-dependent LDR effects using adaptive response experiments in young (7-to 12-week-old) and middle-aged (40-to 62-week-old) C57BL/6 mice. Compared with 3 Gy irradiation, 0.02 Gy preirradiation followed by 3 Gy irradiation prolonged life in young mice but not middle-aged mice. Preirradiation also suppressed irradiation-induced 53BP1 repair foci in the small intestines, splenic apoptosis, and p53 activity in young mice but not middle-aged mice. Young p53+/− C57BL/6 mice did not show these adaptive responses, indicating that insufficient p53 function in young mice mitigated the adaptive responses. Interestingly, p53 activation in middle-aged mice spontaneously became approximately 4.5-fold greater than that in young mice, possibly masking LDR stresses. Furthermore, adaptive responses in young mice, but not in middle-aged mice, suppressed some senescence-associated secretory phenotype (SASP) factors (IL-6, CCL2, CCL5, CXCL1). Thus, LDR-induced adaptive responses associated with specific SASP factors may be attenuated by a combination of reduced DNA damage sensor/transducer function and chronic p53 activation in middle-aged mice.

TMI-Based Dose-Escalated Bone Marrow Transplantation Can Help Preserve the Bone Marrow Microenvironment and Reduce Cellular Senescence in Old Mice

Introduction:Senescence is one of the hallmarks of aging and age-related conditions, including but not limited to, inflammaging, frailty, and various age-related diseases. It has been well-established that radiation and chemotherapy can accelerate senescence. While myeloablative conditioning-based allogeneic hematopoietic cell transplant (HCT) is a curative treatment for patients with leukemia. The dose of total body irradiation (TBI) conditioning has to be reduced in older patients to reduce potential high treatment-related mortality, which may still cause accelerated senescence in aged individuals, resulting in poor survival outcomes. We initiated the testing of a novel total marrow irradiation (TMI) based myeloablative conditioning in older patients to enhance the anti-leukemia effect and overall survival. We hypothesize that this new radiation preconditioning strategy may also reduce radiation-induced senescence in aged individuals compared to TBI to reduce treatment-associated side effects and improve the outcomes of allogeneic HCT. We tested this hypothesis in the present study and further investigated the mechanisms of the effects of TMI-based myeloablative and dose-escalated bone marrow transplantation (BMT) on old mice by examining the changes in expression of various senescence markers and senescence-associated secretory phenotype (SASP) in the bone marrow (BM) and selected organs. Method: The radiation treatment regimens were: TMI (12:4), TMI (16:4) and TBI (12:12), and TBI (16:16), were developed as described (Zuro et al., 2021); i.e. (12:4) means the radiation dose to the BM was 12 Gy and the dose to the organs was 4 Gy. The young (8 weeks old) and aged mice (90 weeks old) of host C57BL/6j (JAX000664, CD45.2) were irradiated in 2 fractions 24h apart, and 25 million whole BM cells from donor (JAX002014, CD45.1) were transplanted by i.v. at 24 h after irradiation. For the hematopoietic stem and progenitor cell (HSPC) population study, more mature hematopoietic progenitors (Lin − Sca-1 −c-Kit + or LK cells) and HSPCs (Lin − Sca-1 + c-Kit + or LSK cells) were examined in BM by flow analysis. Cultured bone marrow mesenchymal stem cells (MSCs) were analyzed for the gene expression of the senescence marker CDNK1a (or p21) and SASP factors MMP13 and CXCL12 by qPCR. For senescence study in organs such as the liver, gut, and spleen, senescence-associated beta-galactosidase (SA-β-gal) staining was performed and expression of the CDNK2a (or p16) was analyzed by qPCR. Results: TMI or TBI treatment with young donor BMT repopulated the BM HSPC population without significant bone damage in aged mice (data not shown). While the BM LK and LSK populations significantly reduced in aged mice after TMI or TBI with young donor BMT compared to aged control mice ( Fig 1A), these populations were not different from young mice groups ( Fig 1B). In addition, TMI or TBI treatment with young donor BMT reduced MSC senescence. Cultured MSCs analysis showed the reduction of p21, MMP13, and CXCL12 expression ( Fig 1C-E) after TMI or TBI with young donor BMT in aged compared to aged control without radiation and BMT. However, while TMI treatment and BMT reduced senescence in the organs, mice receiving TBI treatment and BMT showed a higher senescence burden. In the liver, the aged control mice showed a significant increase in SA-β-gal staining, which was decreased in TMI (12:4) and TMI (16:4) mice ( Fig 1F). In contrast, this effect was not observed in aged mice after TBI (12:12) or (16:16) ( Fig 1G). Moreover, the senescence marker p16 expression in the liver and gut was reduced only in TMI (12:4) and TMI (16:4), not in TBI (12:12) or (16:16) ( Fig 1H, I), whereas the p16 expression in the hematopoietic tissue, spleen was decreased in all treatment groups TMI vs. TBI ( Fig 1J). Conclusion: TMI-based dose escalates with young donor BMT in aged mice preserved BM microenvironment and reduced senescence of organs while TBI treatment causes senescence of the organs. Young donor BMT, which combined with TMI or TBI treatment, preserved bone structure and reduced senescence of HSPC and MSC. However, TMI treatment decreased the SA-β-gal staining and p16 expression significantly in organs such as the liver and gut, but not by TBI treatment. This study showed the senescence reduction in both bone marrow and organs after young donor BMT and TMI treatment. For further study, we have a plan to study the mechanism and signaling pathway of senescence reduction.

Integration of transcriptomes of senescent cell models with multi-tissue patient samples reveals reduced COL6A3 as an inducer of senescence

Senescent cells are a major contributor to age-dependent cardiovascular tissue dysfunction, but knowledge of their invivo cell markers and tissue context is lacking. To reveal tissue-relevant senescence biology, we integrate the transcriptomes of 10 experimental senescence cell models with a 224 multi-tissue gene co-expression network based on RNA-seq data of seven tissues biopsies from ∼600 coronary artery disease (CAD) patients. We identify 56 senescence-associated modules, many enriched in CAD GWAS genes and correlated with cardiometabolic traits-which supports universality of senescence gene programs across tissues and in CAD. Cross-tissue network analyses reveal 86 candidate senescence-associated secretory phenotype (SASP) factors, including COL6A3. Experimental knockdown of COL6A3 induces transcriptional changes that overlap the majority of the experimental senescence models, with cell-cycle arrest linked to modulation of DREAM complex-targeted genes. We provide a transcriptomic resource for cellular senescence and identify candidate biomarkers, SASP factors, and potential drivers of senescence in human tissues.

Transplanting old organs promotes senescence in young recipients

In clinical organ transplantation, donor and recipient ages may differ substantially. Old donor organs accumulate senescent cells that have the capacity to induce senescence in naïve cells. We hypothesized that the engraftment of old organs may induce senescence in younger recipients, promoting age-related pathologies. When performing isogeneic cardiac transplants between age-mismatched C57BL/6 old donor (18 months) mice and young and middle-aged C57BL/6 (3- or 12- month-old) recipients , we observed augmented frequencies of senescent cells in draining lymph nodes, adipose tissue, livers, and hindlimb muscles 30 days after transplantation. These observations went along with compromised physical performance and impaired spatial learning and memory abilities. Systemic levels of the senescence-associated secretory phenotype factors, including mitochondrial DNA (mt-DNA), were elevated in recipients. Of mechanistic relevance, injections of mt-DNA phenocopied effects of age-mismatched organ transplantation on accelerating aging. Single treatment of old donor animals with senolytics prior to transplantation attenuated mt-DNA release and improved physical capacities in young recipients. Collectively, we show that transplanting older organs induces senescence in transplant recipients, resulting in compromised physical and cognitive capacities. Depleting senescent cells with senolytics, in turn, represents a promising approach to improve outcomes of older organs.

Immunomodulatory Effects of Histone Variant H2A.J in Ionizing Radiation Dermatitis

Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.

Removal of gemcitabine-induced senescent cancer cells by targeting glutaminase1 improves the therapeutic effect in pancreatic ductal adenocarcinoma.

Insufficient cancer treatment can induce senescent cancer cell formation and treatment resistance. The characteristics of induced senescent cancer (iSnCa) cells remain unclear. Pancreatic ductal adenocarcinoma (PDAC) has a low and nondurable response rate to current treatments. Our study aimed to analyze the properties of iSnCa cells and the relationship between cellular senescence and prognosis in PDAC. We evaluated the characteristics of gemcitabine-induced senescent cancer cells and the effect of senescence-associated secretory phenotype (SASP) factors released by iSnCa cells on surrounding PDAC cells. The relationship between cellular senescence and the prognosis was investigated in 50 patients with PDAC treated with gemcitabine-based neoadjuvant chemotherapy. Exposure to 5 ng/mL gemcitabine-induced senescence, decreased proliferation and increased senescence-associated β-galactosidase-cell staining without cell death in PDAC cells; the expression of glutaminase1 (GLS1) and SASP factors also increased and caused epithelial-mesenchymal transition in surrounding PDAC cells. iSnCa cells were selectively removed by the GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) through apoptosis induction. Cellular senescence was induced in PDAC cells via insufficient gemcitabine in subcutaneous tumor model mice. GLS1 expression was an independent prognostic factor in patients with PDAC who received gemcitabine-based neoadjuvant chemotherapy. This is the first study to identify the relationship between senescence and GLS1 in PDAC. Low-dose gemcitabine-induced senescence and increased GLS1 expression were observed in PDAC cells. Cellular senescence may contribute to treatment resistance of PDAC, hence targeting GLS1 in iSnCa cells may improve the therapeutic effect.